This invention relates to a flow cytometry system and methods of operating both analytical and drop flow cytometers including apparatus and methods of unclogging or dislodging of media or gas from the inside of devices which may become clogged, Specifically, the invention focuses upon a device which may be used to unclog nozzles, dislocate gas, or clean exterior surfaces of flow cytometers used in the cytometry industry, which are subject to clogging, entrapping gas, or collecting adherents to exterior surfaces.
The desire to unclog, dislocate entrapped gas, and clean nozzles or a nozzle body has been known in some industries for many years. This desire has been quite acute for quite some time in the cytometry field. Cytometers are sometimes used to separate or sort particles from one another based upon the differences detected by shining a high intensity light upon each particle then discerning differences between the amount of light reflected from each particle. These particles may be quite variable based upon the application of the user. They may be biological cells of plant or animal matter or particles of other materials. All of these various particles will vary in size, shape, homogeneity, texture, and adhesion properties.
As shown in FIG. 1, the sheath fluid (8) is forced into the nozzle chamber (11) surrounding the sample tube or sample introduction element (9) at the same time that the sample is injected through the sample tube into the nozzle chamber. Both fluids travel down the chamber with the sample remaining in the center of the chamber. In an ideal sorting-type of setup the sample particles travel in line to the orifice where they exit a microscopic nozzle orifice or nozzle aperture (13) (typically 50-250 microns) into the free fall area within which droplets form and fall. Generally, at this point, the characteristics of the sample are determined at high speeds, sometimes as rapidly as 40,000 drops per second. In the typical application, the samples are then sorted at high speeds into containers based upon the detected characteristics. Thus, predictability and consistency are crucial to accurate detection and to accurate sorting of the samples. Unwelcome variations in the stream emanating from the nozzle, either in volume or in direction or both, may have significant effects on the ability of the user to provide accurate results.
In practice, particles and gas in the sheath fluid or nozzle aperture adherents or the sample particles injected into the nozzle chamber accumulate faster that they can exit the nozzle and can begin filling the nozzle chamber or occluding the nozzle aperture with sample particles or gas bubbles. This accumulation of particles or gas can cause partial or total clogs in the nozzle or nozzle aperture. FIGS. 1, 2 and 3 show a typical cytometer nozzle or exterior surface of a flow cytometer nozzle and area in which such clogging may occur. The problem of clogged nozzles also causes problems relating to the limited shelf life of samples which may be biological in nature and any delays relating to equipment malfunction may require completely starting an experiment from scratch.
Another source of nozzle clogs is related to homogeneity of the sample. The sample preparer, for some reason, may be unable to filter the sample prior to sorting it on the cytometer or the filtered samples may agglutinate. In this case, some of the particles may be larger than the nozzle orifice which will immediately cause clogs.
More specifically, significant advances on sorting sperm for a variety of purposes have been made in recent years. Yet, this type of sample is especially prone to clogging. At present, the only quantitative technique used to achieve the separation of X- and Y-chromosome bearing sperm has been that involving individual discrimination and separation of the sperm through the techniques of flow cytometry. This technique appeared possible as a result of advances and discoveries involving the quantitative dye absorption of X-and Y-chromosome bearing sperm. This was discussed early in U.S. Pat. No. 4,362,246 and significantly expanded upon through the techniques disclosed by Lawrence Johnson in U.S. Pat. No. 5,135,759. The Johnson technique of utilizing flow cytometry to separate X- and Y-chromosome bearing sperm has been so significant an advancement that it has for the first time made the commercial separation of such sperm feasible. While still experimental, separation has been significantly enhanced through the utilization of high speed flow cytometers such as the MoFlo(copyright) flow cytometer produced by Cytomation, Inc. and discussed in a variety of other patents including U.S. Pat. Nos. 5,150,313, 5,602,039, 5,602,349, and 5,643,796 as well as international PCT patent publication WO 96/12171. While the utilization of Cytomation""s MoFlo(copyright) cytometers has permitted great increases in speed, and while these speed increases are particularly relevant given the high number of sperm often used, certain problems have still remained. In spite of the almost ten-fold advances in speed possible by the MoFlo(copyright) flow cytometer, shorter and shorter sorting times have been desired for several reasons. First, it has been discovered that as a practical matter, the sperm are time-critical cells. Their fertility decreases with increased delay time. Second, the collection, sorting, and insemination timings has made speed an item of high commercial importance. Thus, the time critical nature of the sperm cells and the process has made speed an essential element in achieving high efficacy and success rates. Naturally, clogging which greatly increases the time required for sorting of the sperm, can be debilitating to the entire success of the process.
Clogging can also occur from the particular sheath fluid used in cytometry operations. The sheath fluid typically used on a cytometer is that of a saline solution including other additives as needed. This saline may form salt crystals on the outside on the nozzle orifice and slowly restrict the exit orifice of the nozzle or otherwise disturb the natural spraying direction of the nozzle. This may result in a partial or complete clog of the nozzle, and can exacerbate problems caused by sample clumping.
Perhaps one of the most significant problems that those in some fields have faced is that of clearing the orifice of the nozzle without damaging it in some way. While this basic concept seems quite simple, implementation is not so straightforward. The operator or other user was faced with the decision to attempt unclogging in situ while it is attached to the cytometer and properly aligned or to remove the nozzle, clean it and then restart the very tedious and slow realignment steps which are necessary if the nozzle is removed before it can be used again for sorting.
One concept put forward for clearing a nozzle in situ involves inserting a thin wire or similar device into the orifice of the nozzle. While this may seem quite straightforward, the problem of finding the orifice of the nozzle which may be 50 to 250 microns in diameter is quite difficult. Further the small diameter of the wire would make it far too delicate to practically thread into the orifice. Another complication to this approach is the small and difficult-to-reach area where a cytometer nozzle is typically located on the instrument. Even if the aforementioned problems were overcome, the nozzle would likely become damaged as a result of the insertion.
Another concept which has been discussed involves the use of applying a vacuum from the outside of the nozzle orifice, which suffers from a clog or particle. This approach has practical limitations involving sealing around the exterior of the nozzle to allow a vacuum to develop. A vacuum source would need to be generated of sufficient magnitude to be of benefit to the nozzle. This approach is complicated by touching the nozzle in situ and thereby disturbing its sensitive alignment and flow path. Potentially, the vacuum source could make a nozzle clog worse by pulling the particulates inside the nozzle closer and packing them harder into the orifice. Even if a nozzle is unclogged by this method, the operator might be forced to realign the cytometer before using the cytometer.
Another concept used solely in the context of analytical cytometers which do not form droplets is the use of a piezoelectric crystal to set up localized vibrations around the sample injector or sensing aperture for declogging. This concept, shown in U.S. Pat. No. 4,673,288 however, has never been developed or applied for a system compatible with the unique requirements of drop flow cytometry for dislocation of particles. Moreover, the disclosure of using oscillations with respect to analytical flow cytometry teaches the use of pressure change to dislocate gas which teaches away from the disclosed invention.
Another problem particular to cytometry and addressed by this invention is that of trapped air bubbles or gas in the fluidic components of a cytometer. The bubbles can form a compressible medium in the nozzle chamber that affects the precision alignment of the flow stream. In practice,the bubbles are sometimes difficult to remove from the chamber or de-bubble the chamber due, perhaps, to surface tension of the bubble to the walls of the chamber and within the sheath fluid itself. An operator generally is delayed in processing the sample through the cytometer when bubbles are present in the nozzle chamber, thus reducing the efficiency of the process. The cytometer operator previously had no tool to address the problem of trapped gases and had no choice but to wait some amount of time, which would vary greatly, and allow the bubbles to naturally migrate to areas of lesser concern.
Another concern particular to drop flow cytometry systems is in situ cleaning of either the exterior or the interior of the drop flow cytometer nozzle. Adherents which collect on the nozzle disturb the flow path and alignment with the flow path sensing system. Routine cleaning of the nozzle surfaces can maintain the flow path and alignment parameters without having to disassemble the nozzle and clean the parts separate from the drop flow cytometer. Previously, no tool existed to address the problem of cleaning such surfaces of the drop flow cytometer nozzle.
As to both the cytometry industry and the overall desire to unclog nozzles in situ in any industry, the present invention discloses techniques which overcome virtually every one of the previous problems in a practical fashion. Perhaps surprisingly, it satisfies a long-felt need to achieve unclogging, de-bubbling and cleaning of a nozzle in situ with little or no damage to the nozzle and without significantly disturbing it in any way. It is perhaps surprising to those involved in the use of cytometers to see how the problem could be solved in a safe, economical manner without requiring the removal of nozzles.
The present invention includes a variety of aspects which may be selected in different combinations to suit the needs of the user. First, it can function as a nozzle unclogging tool or cleaning tool for a cytometer nozzle or any nozzle which has been partially clogged. Second, it can be used as an unclogging tool for a cytometer nozzle or any nozzle which has become fully clogged or has a particle entrapped. In a basic form, the concept involves taking a source of vibratory energy or using an energy converter which creates mechanical oscillations (as selected with a oscillation selection element or by sweeping a range of oscillations), and with a media or oscillation coupling element present (such as a fluid, gel or other appropriate media) or through a mechanical couple, and transferring the energy from the source and presenting (coupling) it to the problem area or to allow the media in cooperation with the vibration (either ultrasonic or subsonic) to dislodge any matter in the nozzle which may be causing the clog. Such a technique offers advantages to the operator of the cytometer in allowing machine downtime and any resulting realignment to be greatly reduced, thereby making the operation of a cytometer to be more predictable and increasing the likelihood of successfully sorting a particular sample. Third, it can function as a de-bubbling tool to aid the cytometer operator in removing bubbles from a cytometer. This device excites the nozzle and surrounding fluid in such a way that the air quickly migrates to areas where it can be removed. The invention does this by providing a means for a vibratory chamber of media in which to immerse the nozzle, especially the orifice of the nozzle, into the vibrating media in the chamber while the nozzle is still mounted and fluidically attached or by means of mechanical coupling to the exterior surfaceof the nozzle.
Based upon testing which has occurred to date, it functions particularly well on partially clogged nozzles. It appears to typically unclog these nozzles and allow the cytometer stream to return to its original position, thereby eliminating the need for adjustment, so typical prior to the present invention.
One of the broad objects of the invention is to allow for an unclogging tool or portable member configured so as to mechanically couple mechanical oscillations to the exterior of the nozzle to be used while the nozzle is present on the cytometer, in situ. Thus, one goal includes making the device small enough to fit within the existing spaces surrounding the nozzle on cytometers. Another goal would be to allow a means for the device to be moved into position or have a portable member such that the chamber or portable surface configured so as to mechanically couple mechanical oscillations to the exterior of the nozzle is presenting the vibratory media to the nozzle orifice or nozzle exterior and then easily removed from this area such that normal cytometer functions can take place. This goal may be accomplished manually (hand held) such as with a portable device or may be automated with a more permanently mounted unit or automated movement that retracts and engages the nozzle. Likewise, it may be manually activated independent of flow cytometer operation conditions when the operator notices a variation in the processing or it may be automated by sensors detecting a clogging or in response to flow cytometer conditions.
Another goal would be to allow the use of such a device that will not harm the nozzle. A goal for this invention is to unclog the nozzle while preventing any aspects to the procedure which could effect the positioning of the nozzle or the original condition of the nozzle orifice.
Another broad object of the invention is to provide a means of removing air bubbles from the nozzle or debubbling. Some of the goals for this object remain the same as for unclogging in that the device is generally small enough to fit in the space allowed and may be easily moved out of the way after use. A further goal would be to allow the cytometer operator to pull a vacuum on the inside of the nozzle chamber while presenting a surface configured so as to mechanically couple mechanical oscillations to the exterior of the nozzle or a chamber of media to the nozzle and vibrating the media and therefore the nozzle with sufficient energy that any air bubbles in the nozzle will no longer cling to the sides of the nozzle, but will rise to areas where they may removed through fluidic hoses and tubes.
Yet another goal would be to allow for different cytometer nozzles on different cytometer machines to be unclogged and debubbled with this invention. Further, it is a goal to provide this in an economical manner such that it is affordable to the cytometry user.